Project Summary Abstract This proposal strives to elucidate the mechanical determinants of cardiac myocyte (CM) proliferation, maturation, and function. We are focusing on mechanical regulation of CM proliferation because we wish to improve our understanding of why, unlike most cell types, CMs are unable to meaningfully proliferate after they have matured. Interestingly, key cell cycle activators Yap1 and Erk1/2 are regulated by mechanical load in many cell types, but the mechanoregulation of Yap1 and Erk1/2 in CMs is poorly understood. Understanding the mechanical blocks to CM proliferation, as well as mechanical stimuli that promote proliferation, may unlock new strategies of promoting CM proliferation and heart regeneration. Our study will also examine cell cycle activation that results in pathological hypertrophy to identify why true cell division does not occur, and will uncover mechanisms that underly maladaptive remodeling post injury. In the heart, CMs are tightly connected and exert mechanical load on each other. Therefore, maintaining these interactions is essential for meaningful understanding of mechanoregulation of CM functions. We have engineered complex 3D heart tissues that recapitulate these interactions and allow manipulation of specific mechanical forces, including strain, afterload, and matrix stiffness. These tissues are also amenable to live imaging with subcellular resolution. We recently generated multi-colored fluorescent membrane reporter (?brainbow?) vectors and human induced stem cell (hiPSC) lines we will use to examine CM proliferation rates and morphometry when specific mechanical stimuli are applied. In addition, we will study the effects of extracellular matrix (ECM) levels on CM proliferation and morphometry in transgenic mouse models with tunable ECM in vivo. Because the Yap1 transcriptional-coactivator and Erk1/2 have been implicated as key intermediates between mechanical force sensing and gene expression regulation in non-CMs, we will examine how Yap1 and Erk1/2 nuclear localization and target gene expression are modulated in CMs under various mechanical loads in 3D engineered heart tissues in vitro and in in vivo mouse models. The proposed studies are essential for understanding how mechanical strain, afterload, and stiffness regulate key cell cycle mechanotransducers in in vivo models and tissue engineering approaches that will overcome decades of research confounded by broad-acting injuries or in vitro artefacts from 2D cultures.